Biomedical Engineering Reference
In-Depth Information
ferent neuronal tissues in a variety of species from lower verte-
brates to mammals
(39-45)
. This technique utilizes a “bolus”
injection of a membrane-permeant Ca
2
+
indicator dye into the
extracellular space. Briefly, a pipette containing the AM form of
an indicator dye is inserted into the tissue of interest and approx-
imately 400 fl of the dye-containing solution is pressure-ejected
at the desired depth. We typically use patch-like pipettes with a
pipette resistance of 6-9 MOhm when filled with the pipette solu-
tion
(38)
. For the detailed step-by-step description of the tech-
nique (including the list of required equipment as well as tip and
troubleshooting table), we refer the reader to Garaschuk et al.
(112)
. When injected into the brain parenchyma, the dye diffuses
into the cells of interest where it is hydrolyzed by intracellular
esterases. The remaining dye is rapidly removed from the extra-
cellular space by microcirculation
(46)
. The technique accommo-
dates any AM indicator dye of interest (also Ca
2
+
insensitive dyes
like, for example, Calcein AM). Ca
2
+
indicator dyes successfully
used so far include: Fura-2 AM, Fura-PE3 AM, Fura Red AM,
Indo-1 AM, Calcium Green-1 AM, Oregon Green 488 BAPTA-
1 AM, Fluo-4 AM, and Magnesium Green AM.
Figure 3.7
illustrates Ca
2
+
transients evoked in layer 2/3 corti-
cal neurons in response to sensory stimulation. In
A
, neurons in
the barrel cortex were activated by the air-puff induced movement
of the majority of whiskers on the contralateral side of the mouse's
snout. In
B
, cells in the primary visual cortex were activated by
repetitive light flashes. Note that not all cells in
B
are responding
to this kind of stimulus (compare cells 1, 4 with cells 2, 3). The
figure also illustrates two different approaches used for record-
ing stimulus-induced Ca
2
+
transients. In
A
,Ca
2
+
transients were
recorded at 200 Hz sampling rate using line scan mode. While
providing “real time” temporal resolution, this technique reduces
the spatial dimension of the recordings to a single line (
scan line
in
A
). In
B
, the area containing 4 neighboring neurons was imaged
at frame rate of
2.2.1. Example Results
20 Hz. Note that although the individual cell
bodies can be clearly distinguished, the multi cell bolus loading
technique does not provide imaging contrast sufficient to resolve
individual dendritic processes.
∼
∼
2.3. Population
Signals from
Olfactory Receptor
Neuron Terminals in
the Olfactory Bulb
Glomeruli (Fig. 3.3,
Right Panel)
A very large number (
10,000) of olfactory receptor neurons
send their axons to each glomerulus in the olfactory bulb. These
nerve terminals are small,
m in diameter, far below the spatial
resolution obtainable using wide-field microscopy. Thus, wide-
field imaging from the glomerulus will result in a population sig-
nal, a signal that is the population average of what is happening in
all 10,000 terminals. Selective labeling of olfactory receptor neu-
ron nerve terminals with a calcium-sensitive dye was used to visu-
alize the spatial-temporal patterns in the input from the nose to
∼
1
μ
